The red cell membrane skeleton is thought to be involved in the control of cell shape and deformability, parameters which are of critical importance in allowing red cells to negotiate the narrow passages of the microcirculation. Cellular morphology and deformability are unfavorably altered in old red cells and in certain red cell disorders. Our hypothesis is that these changes are due, at least in part, to alterations in skeletal protein components. We have observed that the ratio of the key skeletal components bands 4.la,b changes dramatically as mouse red cells age in vivo. We will explore the biochemical basis for this apparent conversion of 4.lb to 4.la during aging and also examine the effects of this change on the associations of 4.la,b with the bilayer domain and other skeletal elements. We will also explore the basis for an apparent increase in globin binding to skeletons derived from in vivo-aged mouse red cells. We will also explore whether other skeletal changes occur, and will look for similar alterations in human red cells enriched with respect to age by separation techniques. Recent evidence indicates that the major components of the red cell skeleton, including spectrin, ankyrin, and band 4.1, are also present in a variety of other cell types. This suggests that skeletal changes observed during red cell aging might also be manifested during senescence in other cell types, and thus have broad significance. We will also further characterize an elliptocytosis patient whose membranes are deficient in 4.la,b and the sialoglycoprotein PAS 2, which appears to anchor the skeletal core to the bilayer domain via an association with bands 4.la,b. Preliminary results using freeze fracture electron microscopy demonstrate a 50 percent reduction in E-face IMP density in this patient, suggesting that PAS 2 may be one of the components comprising E-face IMPs. Further studies of this are also planned. Our long range goals are to identify skeletal alterations in old and abnormal red cells, to determine the effects of these changes on the stability of the membrane and to determine how these changes might result in a "signal" for the clearance of senescent red cells.